Modulators of cystic fibrosis transmembrane conductance regulator

ABSTRACT

The present invention relates to compounds of formula IVA, formula IVB, or formula IVC, useful as modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator, compositions thereof, and methods therewith. The present invention also relates to methods of treating diseases using such CFTR modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2008/086562 filed Dec. 12, 2008 entitled “MODULATORS OF CYSTICFIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR,” which in turn claimspriority under 35 U.S.C. §119 to U.S. Provisional Application No.61/013,336, filed Dec. 13, 2007 and entitled “MODULATORS OF CYSTICFIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR,” the entire contents ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

ATP cassette transporters are a family of membrane transporter proteinsthat regulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. They arehomologous membrane proteins that bind and use cellular adenosinetriphosphate (ATP) for their specific activities. Some of thesetransporters were discovered as multidrug resistance proteins (like theMDR1-P glycoprotein, or the multidrug resistance protein, MRP1),defending malignant cancer cells against chemotherapeutic agents. Todate, 48 such transporters have been identified and grouped into 7families based on their sequence identity and function.

One member of the ATP cassette transporters family commonly associatedwith disease is the cAMP/ATP-mediated anion channel, CFTR. CFTR isexpressed in a variety of cells types, including absorptive andsecretory epithelia cells, where it regulates anion flux across themembrane, as well as the activity of other ion channels and proteins. Inepithelia cells, normal functioning of CFTR is critical for themaintenance of electrolyte transport throughout the body, includingrespiratory and digestive tissue. CFTR is composed of approximately 1480amino acids that encode a protein made up of a tandem repeat oftransmembrane domains, each containing six transmembrane helices and anucleotide binding domain. The two transmembrane domains are linked by alarge, polar, regulatory (R)-domain with multiple phosphorylation sitesthat regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, more than 1000 diseasecausing mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease. Another mutation, G551D-CFTR involvesthe replacement of Gly with Asp at position 551.

The mutation in CFTR prevents the nascent protein from foldingcorrectly. This results in the inability of the mutant protein to exitthe ER, and traffic to the plasma membrane. As a result, the number ofchannels present in the membrane is far less than observed in cellsexpressing wild-type CFTR. In addition to impaired trafficking, themutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of mutated CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR and G551D-CFTR, other disease causing mutations in CFTR thatresult in defective trafficking, synthesis, and/or channel gating couldbe up- or down-regulated to alter anion secretion and modify diseaseprogression and/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions, chloride andbicarbonate) represents one element in an important mechanism oftransporting ions and water across the epithelium. The other elementsinclude the epithelial Na⁺ channel, ENaC, Na⁺/2Cl⁻/K⁺ co-transporter,Na⁺—K⁺-ATPase pump and the basolateral membrane K⁺ channels, that areresponsible for the uptake of chloride into the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺-K⁺-ATPase pumpand Cl-channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

Defective bicarbonate transport due to mutations in CFTR is hypothesizedto cause defects in certain secretory functions. See, e.g., “Cysticfibrosis: impaired bicarbonate secretion and mucoviscidosis,” Paul M.Quinton, Lancet 2008; 372: 415-417.

In addition to cystic fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. CFTR regulates chloride and bicarbonate flux across theepithelia of many cells to control fluid movement, proteinsolubilization, mucus viscosity, and enzyme activity. Defects in CFTRcan cause blockage of the airway or ducts in many organs, including theliver and pancreas. Any disease which involves thickening of the mucus,impaired fluid regulation, impaired mucus clearance, or blocked ductsleading to inflammation and tissue destruction could be a candidate forpotentiators.

These include, but are not limited to, chronic obstructive pulmonarydisease (COPD), asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, dry eye disease, and Sjögren's Syndrome.COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

It is believed that mutations in CFTR prevent the nascent protein fromfolding correctly, resulting in the inability of this mutant protein toexit the ER, and traffic to the plasma membrane. As a result,insufficient amounts of the mature protein are present at the plasmamembrane and chloride transport within epithelial tissues issignificantly reduced. In fact, this cellular phenomenon of defective ERprocessing of CFTR by the ER machinery, has been shown to be theunderlying basis not only for CF disease, but for a wide range of otherisolated and inherited diseases. The two ways that the ER machinery canmalfunction is either by loss of coupling to ER export of the proteinsleading to degradation, or by the ER accumulation of thesedefective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp745-751 (1999); Shastry, B. S., et al., Neurochem. International, 43, pp1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp 211-222(2002); Morello, J P et al., TIPS, 21, pp. 466-469 (2000); Bross P., etal., Human Mut., 14, pp. 186-198 (1999)]. The diseases associated withthe first class of ER malfunction are cystic fibrosis (due to misfoldedΔF508-CFTR as discussed above), hereditary emphysema (due toa1-antitrypsin; non Piz variants), hereditary hemochromatosis,hoagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, Mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, hereditary emphysema (due to α1-Antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), Diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome (due to Prp processing defect), infertility, pancreatitis, andliver disease.

Other diseases implicated by a mutation in CFTR include male infertilitycaused by congenital bilateral absence of the vas deferens (CBAVD), mildpulmonary disease, idiopathic pancreatitis, and allergicbronchopulmonary aspergillosis (ABPA). See, “CFTR-opathies: diseasephenotypes associated with cystic fibrosis transmembrane regulator genemutations,” Peader G. Noone and Michael R. Knowles, Respir. Res. 2001,2: 328-332 (incorporated herein by reference).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR. Although there arenumerous causes of diarrhea, the major consequences of diarrhealdiseases, resulting from excessive chloride transport are common to all,and include dehydration, acidosis, impaired growth and death. Acute andchronic diarrheas represent a major medical problem in many areas of theworld. Diarrhea is both a significant factor in malnutrition and theleading cause of death (5,000,000 deaths/year) in children less thanfive years old.

Secretory diarrheas are also a dangerous condition in patients withacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). 16 million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Accordingly, there is a need for modulators of CFTR activity, andcompositions thereof, which can be used to modulate the activity of theCFTR in the cell membrane of a mammal.

There is a need for methods of treating diseases caused by mutation inCFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity. These compounds have the generalformula I:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, R⁴,and Ar¹ are described generally and in classes and subclasses below.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease.

DETAILED DESCRIPTION OF THE INVENTION I. General Description ofCompounds of the Invention

The present invention relates to compounds of formula I useful asmodulators of ABC transporter activity:

or a pharmaceutically acceptable salt thereof, wherein:

Ar¹ is a 5-6 membered aromatic monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is optionally fused to a 5-12 membered monocyclic or bicyclic,aromatic, partially unsaturated, or saturated ring, wherein each ringcontains 0-4 heteroatoms independently selected from nitrogen, oxygen,or sulfur, wherein Ar¹ has m substituents, each independently selectedfrom —WR^(W);

W is a bond or is an optionally substituted C₁-C₆ alkylidene chainwherein up to two methylene units of W are optionally and independentlyreplaced by O, —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—,—NR′CO₂—, —O—, —NR′CONR′—, —C(O)NR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—,—NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—;

Z is —CH—, —CR¹—, or N,

m is 0-5;

k is 0-2;

each of R′ is independently —X—R^(X);

X is a bond or is an optionally substituted C₁-C₆ alkylidene chainwherein up to two methylene units of X are optionally and independentlyreplaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—,—NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—,—SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—;

R^(X) is independently R′, halo, NO₂, CN, CF₃, or OCF₃;

R² is hydrogen;

R³ is hydrogen;

R⁴ is hydrogen or a C₁₋₆ aliphatic group optionally substituted with—X—R^(X);

R′ is independently selected from hydrogen or an optionally substitutedgroup selected from a C₁₋C₈ aliphatic group, a 3-8-membered saturated,partially unsaturated, or fully unsaturated monocyclic ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-12 membered saturated, partially unsaturated, or fully unsaturatedbicyclic ring system having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; or two occurrences of R′ are taken togetherwith the atom(s) to which they are bound to form an optionallysubstituted 3-12 membered saturated, partially unsaturated, or fullyunsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₈ hydrocarbon or bicyclic or tricyclic C₈-C₁₄hydrocarbon that is completely saturated or that contains one or moreunits of unsaturation, but which is not aromatic, that has a singlepoint of attachment to the rest of the molecule wherein any individualring in said bicyclic ring system has 3-7 members. Suitable aliphaticgroups include, but are not limited to, linear or branched, substitutedor unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof suchas (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.Suitable cycloaliphatic groups include cycloalkyl, bicyclic cycloalkyl(e.g., decalin), bridged bicycloalkyl such as norbornyl or[2.2.2]bicyclo-octyl, or bridged tricyclic such as adamantyl.

The term “heteroaliphatic”, as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, phosphorus, or silicon.Heteroaliphatic groups may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and include “heterocycle”,“heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic” groups.

The term “heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or“heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic,or tricyclic ring systems in which one or more ring members is anindependently selected heteroatom. In some embodiments, the“heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic”group has three to fourteen ring members in which one or more ringmembers is a heteroatom independently selected from oxygen, sulfur,nitrogen, or phosphorus, and each ring in the system contains 3 to 7ring members.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloaliphatic” and “haloalkoxy” means aliphatic or alkoxy, asthe case may be, substituted with one or more halo atoms. The term“halogen” or “halo” means F, Cl, Br, or I. Examples of haloaliphaticinclude —CHF₂, —CH₂F, —CF₃, —CF₂—, or perhaloalkyl, such as, —CF₂CF₃.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”. The term“aryl” also refers to heteroaryl ring systems as defined hereinbelow.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group are selected fromhalo; —R^(o); —OR^(o); —SR^(o); 1,2-methylene-dioxy; 1,2-ethylenedioxy;phenyl (Ph) optionally substituted with R^(o); —O(Ph) optionallysubstituted with R^(o); —(CH₂)₁₋₂(Ph), optionally substituted withR^(o); —CH═CH(Ph), optionally substituted with R^(o); —NO₂; —CN;—N(R^(o))₂; —NR^(o)C(O)R^(o); —NR^(o)C(O)N(R^(o))₂; —NR^(o)CO₂R^(o);—NR^(o)NR^(o)C(O)R^(o); —NR^(o)NR^(o)C(O)N(R^(o))₂;—NR^(o)NR^(o)CO₂R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —CO₂R^(o);—C(O)R^(o); —C(O)N(R^(o))₂; —OC(O)N(R^(o))₂; —S(O)₂R^(o); —SO₂N(R^(o))₂;—S(O)R^(o); —NR^(o)SO₂N(R^(o))₂; —NR^(o)SO₂R^(o); —C(═S)N(R^(o))₂;—C(═NH)—N(R^(o))₂; or —(CH₂)₀₋₂NHC(O)R^(o) wherein each independentoccurrence of R^(o) is selected from hydrogen, optionally substitutedC₁₋₆ aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclicring, phenyl, —O(Ph), or —CH₂(Ph), or, notwithstanding the definitionabove, two independent occurrences of R^(o), on the same substituent ordifferent substituents, taken together with the atom(s) to which eachR^(o) group is bound, form a 3-8-membered cycloalkyl, heterocyclyl,aryl, or heteroaryl ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Optional substituents on the aliphaticgroup of R^(o) are selected from NH₂, NH(C₁₋₄aliphatic),N(C₁₋₄aliphatic)₂, halo, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic), NO₂, CN,CO₂H, CO₂(C₁₋₄aliphatic), O(haloC₁₋₄ aliphatic), or haloC₁₋₄aliphatic,wherein each of the foregoing C₁₋₄aliphatic groups of R^(o) isunsubstituted.

An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclicring may contain one or more substituents. Suitable substituents on thesaturated carbon of an aliphatic or heteroaliphatic group, or of anon-aromatic heterocyclic ring are selected from those listed above forthe unsaturated carbon of an aryl or heteroaryl group and additionallyinclude the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═NNHC(O)R*,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independentlyselected from hydrogen or an optionally substituted C₁₋₆ aliphatic.Optional substituents on the aliphatic group of R* are selected fromNH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halo, C₁₋₄ aliphatic, OH,O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄aliphatic), or halo(C₁₋₄aliphatic), wherein each of the foregoingC₁₋₄aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen of a non-aromatic heterocyclicring are selected from —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺,—C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, or—NR⁺SO₂R⁺; wherein R⁺ is hydrogen, an optionally substituted C₁₋₆aliphatic, optionally substituted phenyl, optionally substituted —O(Ph),optionally substituted —CH₂(Ph), optionally substituted —(CH₂)₁₋₂(Ph);optionally substituted —CH═CH(Ph); or an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring having one to four heteroatomsindependently selected from oxygen, nitrogen, or sulfur, or,notwithstanding the definition above, two independent occurrences of R⁺,on the same substituent or different substituents, taken together withthe atom(s) to which each R⁺ group is bound, form a 3-8-memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.Optional substituents on the aliphatic group or the phenyl ring of R⁺are selected from NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halo,C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(halo C₁₋₄ aliphatic), or halo(C₁₋₄ aliphatic), whereineach of the foregoing C₁₋₄aliphatic groups of R⁺ is unsubstituted.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of attachment to the rest of themolecule. The term “spirocycloalkylidene” refers to a carbocyclic ringthat may be fully saturated or have one or more units of unsaturationand has two points of attachment from the same ring carbon atom to therest of the molecule.

As detailed above, in some embodiments, two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein), are takentogether together with the atom(s) to which each variable is bound toform a 3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. Exemplary rings that are formed when two independent occurrencesof R^(o) (or R⁺, or any other variable similarly defined herein) aretaken together with the atom(s) to which each variable is bound include,but are not limited to the following: a) two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein) that arebound to the same atom and are taken together with that atom to form aring, for example, N(R)₂, where both occurrences of R^(o) are takentogether with the nitrogen atom to form a piperidin-1-yl,piperazin-1-yl, or morpholin-4-yl group; and b) two independentoccurrences of R^(o) (or R⁺, or any other variable similarly definedherein) that are bound to different atoms and are taken together withboth of those atoms to form a ring, for example where a phenyl group issubstituted with two occurrences of OR^(o)

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) (or R⁺, or any other variablesimilarly defined herein) are taken together with the atom(s) to whicheach variable is bound and that the examples detailed above are notintended to be limiting.

A substituent bond in, e.g., a bicyclic ring system, as shown below,means that the substituent can be attached to any substitutable ringatom on either ring of the bicyclic ring system:

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. E.g., when R² in compounds of formula I is hydrogen,compounds of formula I may exist as tautomers:

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

3. Description of Exemplary Compounds

In some embodiments of the present invention, Ar¹ is selected from:

wherein ring A₁ is a 5-6 membered aromatic monocyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur; or

A₁ and A₂, together, is an 8-14 membered aromatic, bicyclic or tricyclicaryl ring, wherein each ring contains 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In some embodiments, A₁ is an optionally substituted 6 membered aromaticring having 0-4 heteroatoms, wherein said heteroatom is nitrogen. Insome embodiments, A₁ is an optionally substituted phenyl. Or, A₁ is anoptionally substituted pyridyl, pyrimidinyl, pyrazinyl or triazinyl. Or,A₁ is an optionally substituted pyrazinyl or triazinyl. Or, A₁ is anoptionally substituted pyridyl.

In some embodiments, A₁ is an optionally substituted 5-membered aromaticring having 0-3 heteroatoms, wherein said heteroatom is nitrogen,oxygen, or sulfur. In some embodiments, A₁ is an optionally substituted5-membered aromatic ring having 1-2 nitrogen atoms.

In some embodiments, A₂ is an optionally substituted 6 membered aromaticring having 0-4 heteroatoms, wherein said heteroatom is nitrogen. Insome embodiments, A₂ is an optionally substituted phenyl. Or, A₂ is anoptionally substituted pyridyl, pyrimidinyl, pyrazinyl, or triazinyl.

In some embodiments, A₂ is an optionally substituted 5-membered aromaticring having 0-3 heteroatoms, wherein said heteroatom is nitrogen,oxygen, or sulfur. In some embodiments, A₂ is an optionally substituted5-membered aromatic ring having 1-2 nitrogen atoms. In certainembodiments, A₂ is an optionally substituted pyrrolyl.

In some embodiments, A₂ is an optionally substituted 5-7 memberedsaturated or unsaturated heterocyclic ring having 1-3 heteroatomsindependently selected from nitrogen, sulfur, or oxygen. Exemplary suchrings include piperidyl, piperazyl, morpholinyl, thiomorpholinyl,pyrrolidinyl, tetrahydrofuranyl, etc.

In some embodiments, A₂ is an optionally substituted 5-10 memberedsaturated or unsaturated carbocyclic ring. In one embodiment, A₂ is anoptionally substituted 5-10 membered saturated carbocyclic ring.Exemplary such rings include cyclohexyl, cyclopentyl, etc.

In some embodiments, ring A₂ is selected from:

wherein ring A₂ is fused to ring A₁ through two adjacent ring atoms.

In other embodiments, W is a bond or is an optionally substituted C₁-C₆alkylidene chain wherein up to two methylene units of W are optionallyand independently replaced by O, —CO—, —CS—, —COCO—, —CONR′—,—CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —C(O)NR′—,—OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO, —SO₂—, —NR′—,—SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—, and R^(W) is R′ or halo

In still other embodiments, each occurrence of WR^(W) is independently—C1-C3 alkyl, t-butyl, C1-C3 perhaloalkyl, —OH, —O(C1-C3alkyl), —CF₃,—OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R′)(R′),—O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionallysubstituted 5-7 membered heterocylic ring, optionally substituted 5-7membered cycloaliphatic group, optionally substituted monocyclic orbicyclic aromatic ring, optionally substituted arylsulfone, optionallysubstituted 5-membered heteroaryl ring, —N(R′)(R′), —(CH₂)₂N(R′)(R′),—C≡CCH₂N(R′)(R′) or —(CH₂)N(R′)(R′).

In one embodiment of Ar¹ in formula a-i, ring A₁ is a phenyl ring, m is2, and each WR^(W) is independently —CF₃, or optionally substituted 5-7membered heterocylic ring.

In one embodiment of Ar¹ in formula a-i, ring A₁ is a phenyl ring, m is3, and each WR^(W) is independently —OH, or t-butyl.

In one embodiment of Ar¹ in formula a-i, ring A₁ is a phenyl ring, m is2 or 3, and each WR^(W) is independently —OH, —CF₃, or optionallysubstituted 5-7 membered cycloaliphatic group.

In one embodiment of Ar¹ in formula a-i, ring A₁ is a phenyl ring, m is2 or 3, and each WR^(W) is independently —OH, —F, or optionallysubstituted 5-7 membered cycloaliphatic group.

In some embodiments, m is 0. Or, m is 1. Or, m is 2. In someembodiments, m is 3. In yet other embodiments, m is 4.

In one embodiment of the present invention, R¹, R², R³, and R⁴ aresimultaneously hydrogen.

In another embodiment of the present invention, k is 1 or 2 and each R¹is independently C1-C3 alkyl.

In one embodiment, k is 1 and R¹ is C1-C3 alkyl.

In one embodiment, k is 1 and R¹ is methyl.

In one embodiment, k is 1 and R¹ is ethyl.

In one embodiment, k is 1 and R¹ is halo.

In one embodiment, k is 1 and R¹ is CF₃.

In some embodiments, X is a bond or is an optionally substituted C₁₋₆alkylidene chain wherein one or two non-adjacent methylene units areoptionally and independently replaced by O, NR′, S, SO₂, or COO, CO, andR^(X) is R′ or halo. In still other embodiments, each occurrence ofXR^(X) is independently —C₁₋₃alkyl, —O(C₁₋₃alkyl), —CF₃, —OCF₃, —SCF₃,—F, —Cl, —Br, OH, —COOR′, —COR′, —O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′),—CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionally substituted phenyl,—N(R′)(R′), —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′).

In one embodiment, R¹ is H, C1-C4 aliphatic, halo, or C3-C6cycloaliphatic.

In some embodiments, R⁴ is hydrogen. In certain other embodiment, R⁴ isC₁₋₄ straight or branched aliphatic.

In some embodiments, R^(W) is selected from halo, cyano, CF₃, CHF₂,OCHF₂, Me, Et, CH(Me)₂, CHMeEt, n-propyl, t-butyl, —OH, OMe, OEt, OPh,O-fluorophenyl, O-difluorophenyl, O-methoxyphenyl, O-tolyl, O-benzyl,SMe, SCF₃, SCHF₂, SEt, CH₂CN, NH₂, NHMe, N(Me)₂, NHEt, N(Et)₂, C(O)CH₃,C(O)Ph, C(O)NH₂, SPh, SO₂-(amino-pyridyl), SO₂NH₂, SO₂Ph, SO₂NHPh,SO₂—N-morpholino, SO₂—N-pyrrolidyl, N-pyrrolyl, N-morpholino,1-piperidyl, phenyl, benzyl, (cyclohexyl-methylamino)methyl,4-Methyl-2,4-dihydro-pyrazol-3-one-2-yl, benzimidazol-2yl, furan-2-yl,4-methyl-4H-[1,2,4]triazol-3-yl,3-(4′-chlorophenyl)-[1,2,4]oxadiazol-5-yl, NHC(O)Me, NHC(O)Et, NHC(O)Ph,NHSO₂Me, 2-indolyl, 5-indolyl, —CH₂CH₂OH, —OCF₃, O-(2,3-dimethylphenyl),5-methylfuryl, —SO₂—N-piperidyl, 2-tolyl, 3-tolyl, 4-tolyl, O-butyl,NHCO₂C(Me)₃, CO₂C(Me)₃, isopropenyl, n-butyl, O-(2,4-dichlorophenyl),NHSO₂PhMe, O-(3-chloro-5-trifluoromethyl-2-pyridyl),phenylhydroxymethyl, 2-methylpyrrolyl, 3-fluoropyrrolyl,3,3-difluoropyrrolyl, 3,3-dimethylpyrrolyl, 2,5-dimethylpyrrolyl,NHCOCH₂C(Me)₃, O-(2-tert-butyl)phenyl, 2,3-dimethylphenyl,3,4-dimethylphenyl, 4-hydroxymethyl phenyl, 4-dimethylaminophenyl,2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 4-cyanomethylphenyl, 4-isobutylphenyl,3-pyridyl, 4-pyridyl, 4-isopropylphenyl, 3-isopropylphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,3,4-methylenedioxyphenyl, 2-ethoxyphenyl, 3-ethoxyphenyl,4-ethoxyphenyl, 2-methylthiophenyl, 4-methylthiophenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 2,6-dimethoxyphenyl,3,4-dimethoxyphenyl, 5-chloro-2-methoxyphenyl, 2-OCF₃-phenyl,3-trifluoromethoxy-phenyl, 4-trifluoromethoxyphenyl, 2-phenoxyphenyl,4-phenoxyphenyl, 2-fluoro-3-methoxy-phenyl, 2,4-dimethoxy-5-pyrimidyl,5-isopropyl-2-methoxyphenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 3-cyanophenyl, 3-chlorophenyl, 4-chlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 3-chloro-4-fluoro-phenyl,3,5-dichlorophenyl, 2,5-dichlorophenyl, 2,3-dichlorophenyl,3,4-dichlorophenyl, 2,4-dichlorophenyl, 3-methoxycarbonylphenyl,4-methoxycarbonyl phenyl, 3-isopropyloxycarbonylphenyl,3-acetamidophenyl, 4-fluoro-3-methylphenyl, 4-methanesulfinyl-phenyl,4-methanesulfonyl-phenyl, 4-N-(2-N,N-dimethylaminoethyl)carbamoylphenyl,5-acetyl-2-thienyl, 2-benzothienyl, 3-benzothienyl, furan-3-yl,4-methyl-2-thienyl, 5-cyano-2-thienyl, N′-phenylcarbonyl-N-piperazinyl,—NHCO₂Et, —NHCO₂Me, N-pyrrolidinyl, —NHSO₂(CH₂)₂N-piperidine,—NHSO₂(CH₂)₂ N-morpholine, —NHSO₂(CH₂)₂N(Me)₂, COCH₂N(Me)COCH₂NHMe,—CO₂Et, O-propyl, —CH₂CH₂NHCO₂C(Me)₃, aminomethyl, pentyl, adamantyl,cyclopentyl, ethoxyethyl, C(Me)₂CH₂OH, C(Me)₂CO₂Et, —CHOHMe, CH₂CO₂Et,—C(Me)₂CH₂NHCO₂C(Me)₃, O(CH₂)₂OEt, O(CH₂)₂OH, CO₂Me, hydroxymethyl,1-methyl-1-cyclohexyl, 1-methyl-1-cyclooctyl, 1-methyl-1-cycloheptyl,C(Et)₂C(Me)₃, C(Et)₃, CONHCH₂CH(Me)₂, 2-aminomethyl-phenyl, ethenyl,1-piperidinylcarbonyl, ethynyl, cyclohexyl, 4-methylpiperidinyl,—OCO₂Me, —C(Me)₂CH₂NHCO₂CH₂CH(Me)₂, —C(Me)₂CH₂NHCO₂CH₂CH₂CH₃,—C(Me)₂CH₂NHCO₂Et, —C(Me)₂CH₂NHCO₂Me, —C(Me)₂CH₂NHCO₂CH₂C(Me)₃,—CH₂NHCOCF₃, —CH₂NHCO₂C(Me)₃, —C(Me)₂CH₂NHCO₂(CH₂)₃CH₃,C(Me)₂CH₂NHCO₂(CH₂)₂OMe, C(OH)(CF₃)₂,—C(Me)₂CH₂NHCO₂CH₂-tetrahydrofurane-3-yl, C(Me)₂CH₂—O—(CH₂)₂OMe, or3-ethyl-2,6-dioxopiperidin-3-yl.

In one embodiment, R′ is hydrogen.

In one embodiment, R′ is a C1-C8 aliphatic group, optionally substitutedwith up to 3 substituents selected from halo, CN, CF₃, CHF₂, OCF₃, orOCHF₂, wherein up to two methylene units of said C1-C8 aliphatic isoptionally replaced with —CO—, —CONH(C1-C4 alkyl)-, —CO₂—, —OCO—,—N(C1-C4 alkyl)CO₂—, —O—, —N(C1-C4 alkyl)CON(C1-C4 alkyl)-, —OCON(C1-C4alkyl)-, —N(C1-C4 alkyl)CO—, —S—, —N(C1-C4 alkyl)-, —SO₂N(C1-C4 alkyl)-,N(C1-C4 alkyl)SO₂—, or —N(C1-C4 alkyl)SO₂N(C1-C4 alkyl)-.

In one embodiment, R′ is a 3-8 membered saturated, partiallyunsaturated, or fully unsaturated monocyclic ring having 0-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein R′ isoptionally substituted with up to 3 substituents selected from halo, CN,CF₃, CHF₂, OCF₃, OCHF₂, or C1-C6 alkyl, wherein up to two methyleneunits of said C1-C6 alkyl is optionally replaced with —CO—, —CONH(C1-C4alkyl)-, —CO₂—, —OCO—, —N(C1-C4 alkyl)CO₂—, —O—, —N(C1-C4alkyl)CON(C1-C4 alkyl)-, —OCON(C1-C4 alkyl)-, —N(C1-C4 alkyl)CO—, —S—,—N(C1-C4 alkyl)-, —SO₂N(C1-C4 alkyl)-, N(C1-C4 alkyl)SO₂—, or —N(C1-C4alkyl)SO₂N(C1-C4 alkyl)-.

In one embodiment, R′ is an 8-12 membered saturated, partiallyunsaturated, or fully unsaturated bicyclic ring system having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur;wherein R′ is optionally substituted with up to 3 substituents selectedfrom halo, CN, CF₃, CHF₂, OCF₃, OCHF₂, or C1-C6 alkyl, wherein up to twomethylene units of said C1-C6 alkyl is optionally replaced with —CO—,—CONH(C1-C4 alkyl)-, —CO₂—, —OCO—, —N(C1-C4 alkyl)CO₂—, —O—, —N(C1-C4alkyl)CON(C1-C4 alkyl)-, —OCON(C1-C4 alkyl)-, —N(C1-C4 alkyl)CO—, —S—,—N(C1-C4 alkyl)-, —SO₂N(C1-C4 alkyl)-, N(C1-C4 alkyl)SO₂—, or —N(C1-C4alkyl)SO₂N(C1-C4 alkyl)-.

In one embodiment, two occurrences of R′ are taken together with theatom(s) to which they are bound to form an optionally substituted 3-12membered saturated, partially unsaturated, or fully unsaturatedmonocyclic or bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein R′ is optionallysubstituted with up to 3 substituents selected from halo, CN, CF₃, CHF₂,OCF₃, OCHF₂, or C1-C6 alkyl, wherein up to two methylene units of saidC1-C6 alkyl is optionally replaced with —CO—, —CONH(C1-C4 alkyl)-,—CO₂—, —OCO—, —N(C1-C4 alkyl)CO₂—, —O—, —N(C1-C4 alkyl)CON(C1-C4alkyl)-, —OCON(C1-C4 alkyl)-, —N(C1-C4 alkyl)CO—, —S—, —N(C1-C4 alkyl)-,—SO₂N(C1-C4 alkyl)-, N(C1-C4 alkyl)SO₂—, or —N(C1-C4 alkyl)SO₂N(C1-C4alkyl)-.

According to one embodiment, the present invention provides compounds offormula IIA:

According to one embodiment, the present invention provides compounds offormula IIB:

According to one embodiment, the present invention provides compounds offormula IIIA:

wherein each of X₁, X₂, X₃, X₄, and X₅ is independently selected from CHor N.

According to one embodiment, the present invention provides compounds offormula IIIB:

wherein each of X₁, X₂, and X₅ is independently selected from CH or N.

According to one embodiment, the present invention provides compounds offormula IIIC:

wherein each of X₁, X₂, and X₃ is independently selected from CH or N.

According to one embodiment, the present invention provides compounds offormula IIID:

wherein X₅ is independently selected from CH or N and X₆ is O, S, orNR′.

According to one embodiment, the present invention provides compounds offormula IIIE:

wherein X₅ is independently selected from CH or N and X₆ is O, S, orNR′.

In some embodiments of formula IIIA, each of X₁, X₂, X₃, X₄, and X₅ isCH.

In some embodiments of formula IIIA, X₁, X₂, X₃, X₄, and X₅ takentogether is an optionally substituted ring selected from pyridyl,pyrazinyl, or pyrimidinyl.

In some embodiments of formula IIIB, or formula IIIC, X₁, X₂, X₃, or,X₅, taken together with ring A₂ is an optionally substituted ringselected from:

In some embodiments, R^(W) is selected from halo, cyano, CF₃, CHF₂,OCHF₂, Me, Et, CH(Me)₂, CHMeEt, n-propyl, t-butyl, OH, OMe, OEt, OPh,O-fluorophenyl, O-difluorophenyl, O-methoxyphenyl, O-tolyl, O-benzyl,SMe, SCF₃, SCHF₂, SEt, CH₂CN, NH₂, NHMe, N(Me)₂, NHEt, N(Et)₂, C(O)CH₃,C(O)Ph, C(O)NH₂, SPh, SO₂-(amino-pyridyl), SO₂NH₂, SO₂Ph, SO₂NHPh,SO₂—N-morpholino, SO₂—N-pyrrolidyl, N-pyrrolyl, N-morpholino,1-piperidyl, phenyl, benzyl, (cyclohexyl-methylamino)methyl,4-Methyl-2,4-dihydro-pyrazol-3-one-2-yl, benzimidazol-2yl, furan-2-yl,4-methyl-4H-[1,2,4]triazol-3-yl,3-(4′-chlorophenyl)-[1,2,4]oxadiazol-5-yl, NHC(O)Me, NHC(O)Et, NHC(O)Ph,or NHSO₂Me.

In some embodiments, X and R^(X), taken together, is Me, Et, halo, CN,CF₃, OH, OMe, OEt, SO₂N(Me)(fluorophenyl), SO₂-(4-methyl-piperidin-1-yl,or SO₂—N-pyrrolidinyl.

According to another embodiment, the present invention providescompounds of formula IVA:

According to another embodiment, the present invention providescompounds of formula IVB:

According to another embodiment, the present invention providescompounds of formula IVC:

In one embodiment, the present invention provides compounds of formulaIVA, formula IVB, or formula IVC, wherein k is 1, and R¹ is H, Me, Et,or halo. In another embodiment, k is 1 and R¹ is Me. In anotherembodiment, k is 1 and R¹ is Et.

In one embodiment, the present invention provides compounds of formulaIVB, or formula IVC, wherein ring A₂ is an optionally substituted,saturated, unsaturated, or aromatic seven membered ring with 0-3heteroatoms selected from O, S, or N. Exemplary rings include azepanyl,5,5-dimethyl azepanyl, etc.

In one embodiment, the present invention provides compounds of formulaIVB, or formula IVC, wherein ring A₂ is an optionally substituted,saturated, unsaturated, or aromatic six membered ring with 0-3heteroatoms selected from O, S, or N. Exemplary rings includepiperidinyl, 4,4-dimethylpiperidinyl, etc.

In one embodiment, the present invention provides compounds of formulaIVB, or formula IVC, wherein ring A₂ is an optionally substituted,saturated, unsaturated, or aromatic five membered ring with 0-3heteroatoms selected from O, S, or N.

In one embodiment, the present invention provides compounds of formulaIVB, or formula IVC, wherein ring A₂ is an optionally substituted fivemembered ring with one nitrogen atom, e.g., pyrrolyl or pyrrolidinyl.

According to one embodiment of formula IVA, the following compound offormula VA-1 is provided:

wherein each of WR^(W2) and WR^(W4) is independently selected fromhydrogen, CN, CF₃, OCF₃, halo, C1-C6 straight or branched alkyl, 3-12membered cycloaliphatic, phenyl, C5-C10 heteroaryl or C3-C7heterocyclic, wherein said heteroaryl or heterocyclic has up to 3heteroatoms selected from O, S, or N, wherein said WR^(W2) and WR^(W4)is independently and optionally substituted with up to threesubstituents selected from —OR′, —CF₃, —OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃,halo, CN, —COOR′, —COR′, —O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′),—CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, CH₂CN, optionally substitutedphenyl or phenoxy, —N(R′)(R′), —NR′C(O)OR′, —NR′C(O)R′,—(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′); and

WR^(W5) is selected from hydrogen, halo, —OH, NH₂, CN, CHF₂, NHR′,N(R′)₂, —NHC(O)R′, —NHC(O)OR′, NHSO₂R′, —OR′, CH₂OH, CH₂N(R′)₂, C(O)OR′,C(O)N(R′)₂, SO₂NHR′, SO₂N(R′)₂, OSO₂N(R′)₂, OSO₂CF₃, or CH₂NHC(O)OR′.Or, WR^(W4) and WR^(W5) taken together form a 5-7 membered ringcontaining 0-3 three heteroatoms selected from N, O, or S, wherein saidring is optionally substituted with up to three WR^(W) substituents.

In one embodiment, the present invention provides compounds of formulaVA-1, wherein k is 0.

In one embodiment, the present invention provides compounds of formulaVA-1, wherein k is 1 and R¹ is halo.

In one embodiment, the present invention provides compounds of formulaVA-1, wherein k is 1 and R¹ is C1-C3 alkyl.

In one embodiment, the present invention provides compounds of formulaVA-1, wherein k is 1 and R¹ is Me.

In one embodiment, the present invention provides compounds of formulaVA-1, wherein k is 1 and R¹ is ethyl.

In another embodiment, the present invention provides compounds offormula VA-2:

wherein:

ring B is a 5-7 membered monocyclic or bicyclic, heterocyclic orheteroaryl ring optionally substituted with up to n occurrences of-Q-R^(Q);

Q is W;

R^(Q) is R^(W);

m is 0-4;

n is 0-4; and

R¹, k, W, Z, and R^(W) are as defined above.

In one embodiment, m is 0-2. Or, m is 0. Or m is 1.

In one embodiment, n is 0-2. Or, n is 0. Or, n is 1.

In another embodiment, ring B is a 5-7 membered monocyclic, heterocyclicring having up to 2 heteroatoms selected from O, S, or N, optionallysubstituted with up to n occurrences of -Q-R^(Q). Exemplary heterocyclicrings include N-morpholinyl, N-piperidinyl, 4-benzoyl-piperazin-1-yl,pyrrolidin-1-yl, or 4-methyl-piperidin-1-yl.

In another embodiment, ring B is a 5-6 membered monocyclic, heteroarylring having up to 2 heteroatoms selected from O, S, or N, optionallysubstituted with up to n occurrences of -Q-R^(Q). Exemplary such ringsinclude benzimidazol-2-yl, 5-methyl-furan-2-yl,2,5-dimethyl-pyrrol-1-yl, pyridine-4-yl, indol-5-yl, indol-2-yl,2,4-dimethoxy-pyrimidin-5-yl, furan-2-yl, furan-3-yl, 2-acyl-thien-2-yl,benzothiophen-2-yl, 4-methyl-thien-2-yl, 5-cyano-thien-2-yl,3-chloro-5-trifluoromethyl-pyridin-2-yl.

In another embodiment of formula IVA, the present invention providescompounds of formula VA-3:

wherein:

Q is W;

R^(Q) is R^(W);

m is 0-4;

n is 0-4; and

R¹, k, W, Z, and R^(W) are as defined above.

In one embodiment, n is 0-2.

In another embodiment, m is 0-2. In one embodiment, m is 0. In oneembodiment, m is 1. Or, m is 2.

In one embodiment, QR^(Q) taken together is halo, CF₃, OCF₃, CN, C1-C6aliphatic, O—C1-C6 aliphatic, O-phenyl, NH(C1-C6 aliphatic), or N(C1-C6aliphatic)₂, wherein said aliphatic and phenyl are optionallysubstituted with up to three substituents selected from C1-C6 alkyl,O—C1-C6 alkyl, halo, cyano, OH, or CF₃, wherein up to two methyleneunits of said C1-C6 aliphatic or C1-C6 alkyl is optionally replaced with—CO—, —CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—,—NR′CO—, —S—, —NR′—, SOR′, SO₂R′, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—. Inanother embodiment, R′ above is C1-C4 alkyl.

Exemplary QR^(Q) include methyl, isopropyl, sec-butyl, hydroxymethyl,CF₃, NMe₂, CN, CH₂CN, fluoro, chloro, OEt, OMe, SMe, OCF₃, OPh, C(O)OMe,C(O)O-iPr, S(O)Me, NHC(O)Me, or S(O)₂Me.

In another embodiment, the present invention provides compounds offormula VB-1:

wherein:

R^(W1) is hydrogen or C1-C6 aliphatic;

each of R^(W3) is hydrogen or C1-C6 aliphatic; or

both R^(W3) taken together form a C3-C6 cycloalkyl or heterocyclic ringhaving up to two heteroatoms selected from O, S, or NR′, wherein saidring is optionally substituted with up to two WR^(W) substituents;

m is 0-4; and

k, R¹, W, Z, and R^(W) are as defined above.

In one embodiment, WR^(W1) is hydrogen, C1-C6 aliphatic, C(O)C1-C6aliphatic, or C(O)OC1-C6 aliphatic.

In another embodiment, each R^(W3) is hydrogen, C1-C4 alkyl. Or, bothR^(W3) taken together form a C3-C6 cycloaliphatic ring or 5-7 memberedheterocyclic ring having up to two heteroatoms selected from O, S, or N,wherein said cycloaliphatic or heterocyclic ring is optionallysubstituted with up to three substitutents selected from WR^(W1).Exemplary such rings include cyclopropyl, cyclopentyl, optionallysubstituted piperidyl, etc.

In another embodiment, the present invention provides compounds offormula VB-2:

wherein:

ring A₂ is a phenyl or a 5-6 membered heteroaryl ring, wherein ring A₂and the phenyl ring fused thereto together have up 4 substituentsindependently selected from WR^(W);

m is 0-4; and

W, R^(W), Z, k, and R¹ are as defined above.

In one embodiment, ring A₂ is an optionally substituted 5-membered ringselected from pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, thiadiazolyl, oxadiazolyl, or triazolyl.

In one embodiment, ring A₂ is an optionally substituted 5-membered ringselected from pyrrolyl, pyrazolyl, thiadiazolyl, imidazolyl, oxazolyl,or triazolyl. Exemplary such rings include:

wherein said ring is optionally substituted as set forth above.

In another embodiment, ring A₂ is an optionally substituted 6-memberedring. Exemplary such rings include pyridyl, pyrazinyl, or triazinyl. Inanother embodiment, said ring is an optionally pyridyl.

In one embodiment, ring A₂ is phenyl.

In another embodiment, ring A₂ is pyrrolyl, pyrazolyl, pyridyl, orthiadiazolyl.

Exemplary W in formula VB-2 includes a bond, C(O), C(O)O or C1-C6alkylene.

Exemplary R^(W) in formula VB-2 include cyano, halo, C1-C6 aliphatic,C3-C6 cycloaliphatic, aryl, 5-7 membered heterocyclic ring having up totwo heteroatoms selected from O, S, or N, wherein said aliphatic,phenyl, and heterocyclic are independently and optionally substitutedwith up to three substituents selected from C1-C6 alkyl, O—C1-C6 alkyl,halo, cyano, OH, or CF₃, wherein up to two methylene units of said C1-C6aliphatic or C1-C6 alkyl is optionally replaced with —CO—, —CONR′—,—CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′CO—, —S—, —NR′—,—SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—. In another embodiment, R′ above isC1-C4 alkyl.

In one embodiment, the present invention provides compounds of formulaVB-3:

wherein:

G₄ is hydrogen, halo, CN, CF₃, CHF₂, CH₂F, optionally substituted C1-C6aliphatic, aryl-C1-C6 alkyl, or a phenyl, wherein G₄ is optionallysubstituted with up to 4 WR^(W) substituents; wherein up to twomethylene units of said C1-C6 aliphatic or C1-C6 alkyl is optionallyreplaced with —CO—, —CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—,—OCONR′—, —NR′CO—, —S—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—;

G₅ is hydrogen, an optionally substituted C1-C6 aliphatic, CF₃, or CN;wherein said indole ring system is further optionally substituted withup to 3 substituents independently selected from WR^(W).

In one embodiment, G₄ is hydrogen. Or, G₅ is hydrogen.

In another embodiment, G₄ is hydrogen, and G₅ is C1-C6 aliphatic, CF₃,or CN, wherein said aliphatic is optionally substituted with C1-C6alkyl, halo, cyano, or CF₃, and wherein up to two methylene units ofsaid C1-C6 aliphatic or C1-C6 alkyl is optionally replaced with —CO—,—CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′CO—,—S—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—. In another embodiment, R′above is C1-C4 alkyl.

In another embodiment, G₄ is hydrogen, and G₅ is cyano, CF₃, methyl,ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, cyanomethyl,methoxyethyl, CH₂C(O)OMe, (CH₂)₂—NHC(O)O-tert-butyl, or cyclopentyl.

In another embodiment, G₅ is hydrogen, and G₄ is halo, C1-C6 aliphaticor phenyl, wherein said aliphatic or phenyl is optionally substitutedwith C1-C6 alkyl, halo, cyano, or CF₃, wherein up to two methylene unitsof said C1-C6 aliphatic or C1-C6 alkyl is optionally replaced with —CO—,—CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′CO—,—S—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—. In another embodiment, R′above is C1-C4 alkyl.

In another embodiment, G₅ is hydrogen, and G₄ is halo, CF₃,ethoxycarbonyl, t-butyl, 2-methoxyphenyl, 2-ethoxyphenyl,(4-C(O)NH(CH₂)₂—NMe₂)-phenyl, 2-methoxy-4-chloro-phenyl, pyridine-3-yl,4-isopropylphenyl, 2,6-dimethoxyphenyl, sec-butylaminocarbonyl, ethyl,t-butyl, or piperidin-1-ylcarbonyl.

In another embodiment, G₄ and G₅ are both hydrogen, and the nitrogenring atom of said indole ring is substituted with C1-C6 aliphatic,C(O)(C1-C6 aliphatic), or benzyl, wherein said aliphatic or benzyl isoptionally substituted with C1-C6 alkyl, halo, cyano, or CF₃, wherein upto two methylene units of said C1-C6 aliphatic or C1-C6 alkyl isoptionally replaced with —CO—, —CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—,—NR′CONR′—, —OCONR′—, —NR′CO—, —S—, —NR′—, —SO₂NR′—, NR′SO₂—, or—NR′SO₂NR′—. In another embodiment, R′ above is C1-C4 alkyl.

In another embodiment, G₄ and G₅ are both hydrogen, and the nitrogenring atom of said indole ring is substituted with acyl, benzyl,C(O)CH₂N(Me)C(O)CH₂NHMe, or ethoxycarbonyl.

Representative compounds of the present invention are set forth below inTable 1 below.

TABLE 1  1  2

 3  4

 5  6

 7  8

 9 10

11 12

13 14

15 16

17 18

19

4. General Synthetic Schemes

Compounds of the present invention are readily prepared by methods knownin the art. Illustrated in the Examples hereinbelow are exemplarymethods for the preparation of compounds of the present invention.

5. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

In one aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise any ofthe compounds as described herein, and optionally comprise apharmaceutically acceptable carrier, adjuvant or vehicle. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need thereof is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, edisylate(ethanedisulfonate), ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating, or lessening the severity of a condition, disease, or disorderimplicated by CFTR mutation. In certain embodiments, the presentinvention provides a method of treating a condition, disease, ordisorder implicated by a deficiency of the CFTR activity, the methodcomprising administering a composition comprising a compound of formula(I) to a subject, preferably a mammal, in need thereof.

In certain embodiments, the present invention provides a method oftreating cystic fibrosis, asthma, smoke induced COPD, chronicbronchitis, rhinosinusitis, constipation, pancreatitis, pancreaticinsufficiency, male infertility caused by congenital bilateral absenceof the vas deferens (CBAVD), mild pulmonary disease, idiopathicpancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liverdisease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, comprising the step of administering to said mammal aneffective amount of a composition comprising a compound of the presentinvention.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of the present invention.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of the diseases,disorders or conditions as recited above.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of the diseases, disorders or conditions as recited above.

In certain embodiments, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain genotypes exhibiting residual CFTRactivity, e.g., class III mutations (impaired regulation or gating),class IV mutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Transmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic insufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or patch), bucally, as an oral or nasal spray,or the like, depending on the severity of the infection being treated.In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.01 mg/kgto about 50 mg/kg and preferably from about 0.5 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The activity of a compound utilized in this invention as a modulator ofCFTR may be assayed according to methods described generally in the artand in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated.”

In one embodiment, the additional agent is selected from a mucolyticagent, a bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent. In a further embodiment, theadditional agent is a CFTR modulator other than a compound of thepresent invention.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating CFTR activity in abiological sample or a patient (e.g., in vitro or in vivo), which methodcomprises administering to the patient, or contacting said biologicalsample with a compound of Formula (I) or a composition comprising saidcompound. The term “biological sample”, as used herein, includes,without limitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Modulation of CFTR in a biological sample is useful for a variety ofpurposes that are known to one of skill in the art. Examples of suchpurposes include, but are not limited to, the study of CFTR inbiological and pathological phenomena; and the comparative evaluation ofnew modulators of CFTR.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formula (I). In preferredembodiments, the anion channel is a chloride channel or a bicarbonatechannel. In other preferred embodiments, the anion channel is a chloridechannel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional CFTR in a membrane of acell, comprising the step of contacting said cell with a compound ofFormula (I).

According to another preferred embodiment, the activity of the CFTR ismeasured by measuring the transmembrane voltage potential. Means formeasuring the voltage potential across a membrane in the biologicalsample may employ any of the known methods in the art, such as opticalmembrane potential assay or other electrophysiological methods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells.” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R.Y. Tsien (1997); “Improved indicators of cell membrane potential thatuse fluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of CFTR or a fragment thereof in a biologicalsample in vitro or in vivo comprising (i) a composition comprising acompound of Formula (I) or any of the above embodiments; and (ii)instructions for a) contacting the composition with the biologicalsample and b) measuring activity of said CFTR or a fragment thereof. Inone embodiment, the kit further comprises instructions for a) contactingan additional composition with the biological sample; b) measuring theactivity of said CFTR or a fragment thereof in the presence of saidadditional compound, and c) comparing the activity of the CFTR in thepresence of the additional compound with the density of the CFTR in thepresence of a composition of Formula (I). In preferred embodiments, thekit is used to measure the density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES General Synthetic Schemes

Compounds of the present invention are readily prepared by methods knownin the art. Illustrated below are exemplary methods for the preparationof compounds of the present invention.

The schemes below illustrate the synthesis compounds of Formula (I) ofthe present invention.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example 1 Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide(Compound 14, Table 1)

Ethyl 4-hydroxy-2-methylpyrimidine-5-carboxylate (1.5 g, 8.234 mmol) wassuspended in DME (48 mL) and TBAF (13.17 mL, 1 M solution in THF, 13.17mmol) was added dropwise at 0° C. The resulting solution was stirred for10 minutes and a solution of 1-bromo-2-butanone (1.318 g, 8.728 mmol) inDME (3 mL) was added dropwise. The reaction was stirred at roomtemperature overnight. The solution was then concentrated in vacuo andpartitioned between EtOAc and saturated aqueous NH₄Cl. The EtOAc layerwas separated, dried over Na₂SO₄ and concentrated in vacuo to give anoil. The crude material was purified by flash chromatography 0-100%EtOAc/hexanes to obtain pure desired product, ethyl2-methyl-6-oxo-1-(2-oxobutyl)-1,6-dihydropyrimidine-5-carboxylate, as anoff white solid; 1.73 g (83%). LC/MS (10-99% CH₃CN/0.05% TFA inH₂O/0.05% TFA gradient over 3 min): M+H m/z 252.9, retention time 0.76minutes. ¹H NMR (400.0 MHz, CDCl₃) δ 8.62 (s, 1H), 4.88 (s, 2H), 4.38(m, 2H), 2.68 (m, 2H), 2.49 (s, 3H), 1.39 (t, J=7.1 Hz, 3H) and 1.18 (t,J=7.3 Hz, 3H).

To a solution of sodium ethoxide in ethanol (1.906 g, 2.196 mL, 5.882mmol) under a nitrogen atmosphere was added ethanol (9.2 mL), yielding a5% w/w sodium ethoxide solution. Ethyl2-methyl-6-oxo-1-(2-oxobutyl)-1,6-dihydropyrimidine-5-carboxylate (0.74g, 2.941 mmol) dissolved in ethanol (7 mL) was added dropwise to thesodium ethoxide solution. The reaction mixture was stirred for 30minutes, the solvent was removed under reduced pressure, and theresulting solid was treated with 6M HCl to pH 5. The resultingprecipitate, ethyl7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxylate, wascollected by vacuum filtration as a tan solid; 0.63 g (92%). LC/MS(10-99% CH₃CN/0.05% TFA in H₂O/0.05% TFA gradient over 3 min): M+H m/z235.1, retention time 0.99 minutes. ¹H NMR (400.0 MHz, DMSO) δ 12.74(bs, 1H), 8.37 (s, 1H), 7.17 (t, J=0.9 Hz, 1H), 6.00 (d, J=1.9 Hz, 1H),4.20 (q, J=7.1 Hz, 2H), 2.57-2.50 (m, 2H), 1.27 (t, J=7.1 Hz, 3H) and1.18 (t, J=7.5 Hz, 3H).

Ethyl 7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxylate(0.63 g, 2.711 mmol) was dissolved in a solution of methanol (8mL)/sodium hydroxide (13.56 mL, 2.0 M, 27.11 mmol) and heated at refluxfor 3 hours. The mixture was allowed to cool to room temperature andmethanol was removed in vacuo. The aqueous solution was cooled to 0° C.and concentrated HCl was slowly added until a precipitate formed (pH 4).The precipitate was filtered, washed with water and dried to give7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxylic acid;0.43 g (77%). LC/MS (10-99% CH₃CN/0.05% TFA in H₂O/0.05% TFA gradientover 3 min): M+H m/z 207.1, retention time 1.10 minutes. ¹H NMR (400.0MHz, DMSO) δ 13.42 (bs, 1H), 12.89 (bs, 1H), 8.51 (s, 1H), 7.29 (t,J=0.8 Hz, 1H), 6.17 (d, J=1.8 Hz, 1H), 2.61-2.50 (m, 2H) and 1.20 (t,J=7.5 Hz, 3H).

To a solution of7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxylic acid(32.3 mg, 0.1566 mmol) in 2-methyltetrahydrofuran (400 μL) was added1-Propanephosphonic acid cyclic anhydride (249.1 mg, 233.0 μL, 0.3915mmol) followed by the addition of pyridine (24.77 mg, 25.33 μL, 0.3132mmol). The reaction was sealed and heated at 45° C. for 30 minutes uponwhich 5-amino-2,4-di-tert-butylphenol (41.59 mg, 0.1879 mmol) was addedand the reaction was heated at 45° C. for 16 h. The solvent was removedin vacuo and the residue was purified by reverse phase HPLC (40-100%acetonitrile with 0.035% TFA in water with 0.05% TFA) to giveN-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide.LC/MS (10-99% CH₃CN/0.05% TFA in H₂O/0.05% TFA gradient over 3 min): M+Hm/z 410.5, retention time 2.24 minutes. ¹H NMR (400.0 MHz, DMSO) δ 13.07(d, J=6.6 Hz, 1H), 10.72 (s, 1H), 9.20 (s, 1H), 8.55 (d, J=6.8 Hz, 1H),7.32 (s, 1H), 7.16 (s, 1H), 7.11 (s, 1H), 6.11 (d, J=1.7 Hz, 1H), 2.59(q, J=7.5 Hz, 2H), 1.36 (s, 18H) and 1.22 (t, J=7.5 Hz, 3H).

Example 2 Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide(Compound 5, Table 1)

A mixture of 1H-pyrazol-5-amine 1 (2 g, 24.1 mmol) and diethyl2-(ethoxymethylene)malonate (10.4 g, 48.2 mmol) was heated at 200° C.until the starting material was consumed completely. The reactionmixture was allowed to cool to room temperature and EtOH (10 mL) wasadded. A precipitate formed, which was removed by filtration, washedwith EtOH and dried under vacuum to give diethyl2-((1H-pyrazol-5-ylamino)methylene)malonate; 4.0 g (66%). ¹H NMR (300MHz, CDCl₃) δ 10.95 (d, J=13.5 Hz, 1H), 8.60 (d, J=13.8 Hz, 1H), 7.52(d, J=2.4 Hz, 1H), 6.08 (d, J=2.4 Hz, 1H), 4.30 (q, J=7.2 Hz, 2H), 4.22(q, J=7.2 Hz, 2H), 1.36 (t, J=7.2 Hz, 3H), 1.31 (t, J=7.2 Hz, 3H).

A solution of the 2-((1H-pyrazol-5-ylamino)methylene)malonate (4.0 g,15.8 mmol) in Dowtherm (15 mL) was heated to 250° C. for 2 hours. Thereaction mixture was allowed to cool to room temperature, DMF/EtOH (10mL, 1/1, v/v) was added to the mixture and formed precipitate wasfiltered, which was washed with EtOH and dried under vacuum to giveethyl 7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate; 1.9 g(58%). ¹H NMR (300 MHz, DMSO) δ 13.01 (brs, 1H), 8.58 (s, 1H), 7.91 (d,J=2.1 Hz, 1H), 6.30 (d, J=1.8 Hz, 1H), 4.22 (q, J=7.2 Hz, 2H), 1.27 (t,J=7.2 Hz, 3H).

A suspension of ethyl7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate (1.9 g, 9.2mmol) in 10% aq. NaOH (12 mL, 30.0 mmol) was refluxed for 2 hours. Thereaction mixture was allowed to cool to room temperature; the solutionwas acidified to pH 3-4 with 6 M HCl. The forming precipitate wasfiltered, washed with water and dried to afford7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic acid; 1.6 g(97%). ¹H NMR (300 MHz, DMSO) δ 8.53 (s, 1H), 7.94 (d, J=1.8 Hz, 1H),6.32 (d, J=1.8 Hz, 1H). MS (ESI) m/z: 177.9 [M−H]⁻.

To a vial charged with7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic acid (50 mg,0.28 mmol) and HATU (117 mg, 0.31 mmol) was added 2 mL of THF followedby triethylamine (85 mg, 0.84 mmol). The reaction mixture was allowed tostir for 10 minutes at room temperature, upon which5-amino-2,4-di-tert-butylphenol (62 mg, 0.28 mmol) was added and thereaction was heated at 65° C. for 16 h. After 16 h, the reaction wascooled to room temperature and the solvent was removed in vacuo. Theresidue was purified by reverse phase HPLC (10%-99% CH₃CN (0.035%TFA)/H₂ 0 (0.05% TFA)). LC/MS (10-99% CH₃CN/0.05% TFA in H₂O/0.05% TFAgradient over 3 min) to giveN-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide;M+H m/z 383.3, retention time 1.71 minutes. ¹H NMR (400.0 MHz, DMSO) δ13.48 (s, 1H), 10.68 (s, 1H), 9.24 (s, 1H), 8.78 (s, 1H), 8.06 (d, J=2.0Hz, 1H), 7.17 (s, 1H), 7.13 (s, 1H), 6.41 (d, J=2.0 Hz, 1H), 1.36 (d,J=4.5 Hz, 18H).

Example 3 Synthesis of 2-amino-5-cyclopentyl-4-hydroxybenzonitrile

To a stirring solution of 2-cyclopentyl phenol (7.9 g, 48.7 mmol) inacetic acid (32 mL) and water (16 mL) was added HBr (33% in AcOH, 50.45mL, 292.2 mmol) followed by the dropwise addition of DMSO (34.8 g, 31.6mL, 445.0 mmol) over 10 min. The reaction was quenched with saturatedaqueous NaHCO₃ and concentrated in vacuo to remove gasses. The residuewas brought up in ether (200 mL), washed with water (2×100 mL) and brine(100 mL) then dried over Na₂SO₄. The solution was filtered andconcentrated in vacuo to produce an oil which was purified by silica gelchromatography (0-10% ethyl acetate/hexane) to provide4-bromo-2-cyclopentylphenol (10.5 g, 89% yield) as a colorless oil. ¹HNMR (400.0 MHz, DMSO-d₆) δ 9.59 (s, 1H), 7.20 (d, J=2.5 Hz, 1H), 7.13(dd, J=2.5, 8.5 Hz, 1H), 6.73 (d, J=8.5 Hz, 1H), 3.21-3.13 (m, 1H),1.95-1.88 (m, 2H), 1.77-1.69 (m, 2H), 1.65-1.44 (m, 4H).

4-Bromo-2-cyclopentylphenol (10.0 g, 41.47 mmol) and DMAP (253 mg, 2.07mmol) was dissolved in dichloromethane (50 mL) and triethylamine (11.6mL, 82.94 mmol), cooled to 0° C. and treated with methyl chloroformate(4.8 mL, 62.20 mmol). The reaction was allowed to warm to roomtemperature over 2 h. The reaction was quenched with water, the layersseparated, and the aqueous layer re-extracted with dichloromethane. Thecombined organic extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo to yield an oil that was purified by silica gelchromatography (20% ethyl acetate/hexane) to yield4-bromo-2-cyclopentylphenyl methyl carbonate (10.5 g, 85% yield). ¹H NMR(400.0 MHz, DMSO-d₆) δ 7.52 (d, J=2.4 Hz, 1H), 7.44 (dd, J=2.4, 8.6 Hz,1H), 7.22-7.17 (m, 1H), 3.84 (s, 3H), 3.07-2.98 (m, 1H), 1.95-1.88 (m,2H), 1.79-1.71 (m, 2H), 1.66-1.46 (m, 4H).

Concentrated H₂SO₄ (115 mL) was added to 4-bromo-2-cyclopentylphenylmethyl carbonate (26.09 g, 87.21 mmol) and the mixture stirred andcooled to −10° C. KNO₃ (13.22 g, 130.80 mmol) was then added in portionswith continuous stirring. The reaction was stirred at −10° C. for 1 hthen quenched with ice resulting in precipitation of an off-white solid.The solid was filtered, washed with water and dried to provide theproduct. The water phase was extracted with dichloromethane (3×10 mL)and the combined organic extracts dried over Na₂SO₄. Purification bysilica gel chromatography (5-20% ethyl acetate/hexane) providedadditional 4-bromo-2-cyclopentyl-5-nitrophenyl methyl carbonate(combined 21.72 g, 72% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 8.12 (s,1H), 7.88 (s, 1H), 3.88 (d, J=5.7 Hz, 3H), 3.13 (dd, J=9.4, 17.2 Hz,1H), 1.96-1.92 (m, 2H), 1.80-1.75 (m, 2H), 1.68-1.54 (m, 4H).

To a microwave vial charged with 4-bromo-2-cyclopentyl-5-nitrophenylmethyl carbonate (102 mg, 0.29 mmol), zinc cyanide (35 mg, 0.30 mmol)and Pd(PPh₃)₄ (21 mg, 0.02 mmol) under an N₂ atmosphere was added DMF(500 μL). The reaction was heated under microwave irradiation at 130° C.for 30 min. The reaction was quenched with saturated aqueous Na₂CO₃ andextracted with ethyl acetate (3×10 mL). The combined organic extractswere dried over Na₂SO₄, filtered and concentrated in vacuo to yield abrown oil. Purification by silica gel chromatography (0-15% ethylacetate/hexanes) afforded 5-cyclopentyl-4-hydroxy-2-nitrobenzonitrile asa light yellow solid (40 mg, 58% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ11.62 (s, 1H), 7.84 (s, 1H), 7.70 (s, 1H), 3.29-3.24 (m, 1H), 1.99-1.93(m, 2H), 1.78-1.76 (m, 2H), 1.66-1.57 (m, 4H).

A flask containing 10% Pd/C (4 mg) was evacuated and placed under a N₂atmosphere and suspended in ethanol (2 mL). To this was added5-cyclopentyl-4-hydroxy-2-nitrobenzonitrile (42 mg, 0.18 mmol) as asolution in ethanol (1.5 mL). The reaction was stirred under H₂atmosphere for 2 h, then filtered and concentrated in vacuo to provide2-amino-5-cyclopentyl-4-hydroxybenzonitrile as a yellow oil (36 mg,quantitative yield). M+H m/z 203.1.

Example 4 Synthesis of 5-amino-2-cyclopentyl-4-methylphenol

To a microwave tube charged with 4-bromo-2-cyclopentyl-5-nitrophenylmethyl carbonate (500 mg, 1.45 mmol), Pd(dppf)Cl₂ (96 mg, 0.13 mmol),potassium trifluoro-methyl-boron (177 mg, 1.45 mmol) and cesiumcarbonate (1420 mg, 4.36 mmol) was added tetrahydrofuran (2.5 mL) andwater (1.25 mL). The reaction heated at 110° C. for 35 min undermicrowave irradiation. The reaction was partitioned between ethylacetate and water. The organic layer was separated, dried over Na₂SO₄,filtered and concentrated in vacuo to yield a brown oil. Purification bysilica gel chromatography (0-6% ethyl acetate/hexanes) provided2-cyclopentyl-4-methyl-5-nitro-phenol (167 mg, 52% yield). ¹H NMR (400.0MHz, DMSO-d₆) δ 10.08 (s, 1H), 7.43-7.38 (m, 1H), 7.22 (s, 1H),3.28-3.21 (m, 1H), 2.43 (s, 3H), 1.96-1.91 (m, 2H), 1.80-1.51 (m, 6H).

A flask charged with 10% Pd/C (16 mg) was evacuated and placed under aN₂ atmosphere. To this was added 2-cyclopentyl-4-methyl-5-nitro-phenol(160 mg, 0.72 mmol) as a solution in methanol (3 mL). The reactionmixture was stirred under H₂ atmosphere for 4 h, then filtered andconcentrated in vacuo to provide 5-amino-2-cyclopentyl-4-methylphenol alight tan solid (130 mg, 94% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 8.47(s, 1H), 6.60 (s, 1H), 6.08 (s, 1H), 4.44 (s, 2H), 3.02 (dd, J=2.4, 17.2Hz, 1H), 1.91 (s, 3H), 1.84-1.77 (m, 2H), 1.71-1.66 (m, 2H), 1.58-1.54(m, 2H), 1.44-1.39 (m, 2H).

Example 5 Synthesis of 4-(pyrrolidin-1-yl)-2-(trifluoromethyl)aniline

To a solution of 4-nitro-3-(trifluoromethyl)aniline (2.0 g, 9.7 mmol) intoluene (30 mL) was added tetrahydrofuran-2,5-dione (1.2 g, 11.6 mmol)and the mixture refluxed for 1.5 h. The reaction mixture was cooled,filtered, and the solid washed with ether to provide4-(4-nitro-3-(trifluoromethyl)phenylamino)-4-oxobutanoic acid (1.1 g,39% yield). M+H m/z 307.3.

A solution of 4-(4-nitro-3-(trifluoromethyl)phenylamino)-4-oxobutanoicacid (1.1 g, 3.6 mmol) and NaOAc (1.6 g, 19.7 mmol) in acetic anhydride(15 mL) was stirred for 16 h at 80° C. The reaction was cooled andfiltered. The filtrate was diluted with water and extracted withdichloromethane. The combined organic layers were washed with 1 N NaOH,dried over MgSO₄, filtered, and concentrated in vacuo to provide1-(4-nitro-3-(trifluoromethyl)phenyl)pyrrolidine-2,5-dione (0.4 g, 39%yield). M+H m/z 289.1.

To a solution of1-(4-nitro-3-(trifluoromethyl)phenyl)pyrrolidine-2,5-dione (400 mg, 1.39mmol) in THF (10 mL) was added BH₃ (1.39 mL of 1 M in THF, 1.39 mmol)dropwise. The reaction mixture was then refluxed under N₂ atmosphere for16 h. The reaction was cooled, quenched with methanol, and concentratedin vacuo to provide 1-(4-nitro-3-(trifluoromethyl)phenyl)pyrrolidine asa yellow solid (360 mg, quantitative yield). M+H m/z 261.1.

To a flask charged with 10% Pd/C (50 mg) under inert atmosphere wasadded a solution of 1-(4-nitro-3-(trifluoromethyl)phenyl)pyrrolidine(350 mg, 1.34 mmol) in ethanol. The reaction was stirred under H₂atmosphere for 16 h, then filtered and dried down to provide4-(pyrrolidin-1-yl)-2-(trifluoromethyl)aniline (300 mg, 97% yield). M+Hm/z 231.3. ¹H NMR (400.0 MHz, DMSO-d₆) δ 6.84 (d, J=8.8 Hz, 1H), 6.72(dd, J=2.5, 8.8 Hz, 1H), 6.53 (d, J=2.7 Hz, 1H), 4.74 (s, 2H), 3.18 (m,4H), 1.99-1.95 (m, 4H).

Set forth below in Table 2 is the characterizing data for compounds ofthe present invention prepared according to the above Examples.

TABLE 2 LC/MS LC/RT Cmpd # M + 1 min NMR 1 397.50 1.74 ¹ H NMR (400 MHz,DMSO) δ 13.32 (s, 1H), 10.72 (s, 1H), 9.24 (s, 1H), 8.72 (s, 1H), 7.16(d, J = 8.0 Hz, 2H),6.25 (s, 1H), 2.35 (s, 3H), 1.37 (s, 9H), 1.36 (s,9H) 2 350.40 1.48 ¹ H NMR (400.0 MHz, DMSO) δ 13.47 (s, 1H), 11.10 (s,1H), 10.75 (s, 1H), 8.79 (s, 1H), 8.07 (d, J = 2 Hz, 1H), 8.06 (d, J =1.7, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.02 (dd, J = 1.8, 8.6 Hz, 1H),6.99(d, J = 2.3 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 1.39 (s, 9H) 3 433.202.10 4 364.30 1.40 ¹ H NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 10.96 (s,1H), 10.80 (s, 1H), 8.75 (s, 1H), 7.58 (d, J = 12.3 Hz,2H), , 7.31 (t, J= 2.7 Hz, 1H), 6.40 (s, 1H), 6.25 (s, 1H),2.36 (s, 3H), 1.44 (s, 9H) 5383.30 1.71 6 350.50 1.35 7 389.30 1.95 ¹ H NMR (400.0 MHz, DMSO) δ13.13 (d, J = 6.8 Hz, 1H), 11.83 (d, J = 2.2 Hz, 1H), 11.36 (s, 1H),8.57 (d, J = 6.8 Hz, 1H), 8.29 (d, J = 1.5 Hz, 1H), 7.91 (s, 1H), 7.54(d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.20-7.17 (m, 1H), 6.13 (d, J = 1.8Hz, 1H), 2.60 (d, J = 7.5 Hz, 2H) and 1.22 (t, J = 7.5 Hz, 3H) 8 362.201.29 9 377.50 2.09 ¹ H NMR (400.0 MHz, DMSO) δ 13.07 (d, J = 6.9 Hz,1H), 11.22 (s, 1H), 10.72 (s, 1H), 8.55 (d, J = 6.7 Hz, 1H), 8.07 (d, J= 1.8 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H), 6.99-6.96 (m,2H), 6.11 (d, J = 1.8 Hz, 1H), 2.63-2.57 (q, 2H), 1.39 (s, 9H)and 1.22(t, J = 7.5Hz, 3H) 10 308.30 2.17 11 389.10 1.72 12 412.20 2.54 13392.30 1.48 14 410.50 2.24 ¹ H NMR (400.0 MHz, DMSO) δ 13.07 (d, J = 6.6Hz, 1H), 10.72 (s, 1H), 9.20 (s, 1H), 8.55 (d, J = 6.8 Hz, 1H), 7.32 (s,1H), 7.16 (s, 1H), 7.11 (s, 1H), 6.11(d, J = 1.7Hz, 1H), 2.59 (q, J =7.5 Hz, 2H), 1.36 (s, 18H) and 1.22 (t, J= 7.5 Hz, 3H) 15 398.20 2.10 ¹H NMR (400.0 MHz, DMSO) δ 13.14 (s, 1H), 11.37 (d, J = 2.2 Hz, 1H), 9.38(s, 1H), 8.54 (s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.30 (s, 1H), 6.99(d, J= 13.8 Hz, 1H), 6.09 (d, J = 1.7 Hz, 1H), 2.21 (s, 3H), 2.12-2.07 (m,2H), 1.60-1.51 (m, 2H), 1.42-1.36 (m, 6H) and 1.25 (s, 3H) 16 420.302.06 ¹ H NMR (400.0 MHz, DMSO) δ 13.14 (s, 1H), 11.40 (s, 1H), 10.28 (s,1H), 8.54 (s, 1H), 7.92 (s, 1H), 7.35 (s, 1H), 7.31 (s, 1H), 6.08 (d, J= 1.7 Hz, 1H), 3.20-3.16 (m, 1H), 2.20 (s, 3H), 1.94 (d, J = 5.0 Hz, 2H)and 1.77-1.51 (m, 6H) 17 375.10 1.70 ¹ H NMR (400.0 MHz, DMSO) δ 13.12(d, J = 6.4 Hz, 1H), 11.83 (d, J = 2.3 Hz, 1H), 11.35 (s, 1H), 8.56 (d,J = 6.8 Hz, 1H), 8.29 (d, J = 1.6 Hz, 1H), 7.91 (t, J = 1.3 Hz, 1H),7.55 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.19 (dd, J = 1.8, 8.6 Hz, 1H),6.09 (d, J = 1.8 Hz, 1H) and 2.22 (s, 3H) 18 366 1.851 ¹ H NMR (400.0MHz, DMSO-d ₆ ) δ 13.07 (s, 1H), 11.02 (s, 1H), 9.08 (s, 1H), 8.53 (s,1H), 7.88 (s, 1H), 7.30 (s, H), 6.94 (s, 1H), 6.07 (d, J = 1.8 Hz, 1H),3.17-3.12 (m, 1H), 2.23 (s, 3H), 2.20 (s, 3H), 1.92-1.86 (m, 2H),1.76-1.73 (m, 2H), 1.62-1.48 (m, 4H). 19 377 1.76

Assays for Detecting and Measuring ΔF508-CFTR Potentiation Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluoresecent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-Free Bath Solution: Chloride Salts in Bath Solution #1 areSubstituted with Gluconate Salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours. Electrophysiological Assays for assayingΔF508-CFTR modulation properties of compounds.

1. Ussing Chamber Assay

Ussing chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that wereprecoated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUssing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,IA). Briefly, HBE were examined under voltage-clamp recording conditions(V_(hold)=0 mV) at 37° C. The basolateral solution contained (in mM) 145NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10 Glucose, 10HEPES (pH adjusted to 7.35 with NaOH) and the apical solution contained(in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, 10 HEPES (pHadjusted to 7.35 with NaOH).

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

2. Patch-Clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mlamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-C1, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(C1) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compounds of the invention are useful as modulators of ATP bindingcassette transporters. Examples of activities and efficacies of thecompounds of the invention are shown below in Table 3. The compoundactivity is illustrated with “+++” if activity was measured to be lessthan 5.0 μM, “++” if activity was measured to be from 5 μM to 20.0 μM,“+” if activity was measured to be greater than 20.0 μM, and “−” if nodata was available. The efficacy is illustrated with “+++” if efficacywas calculated to be greater than 100%, “++” if efficacy was calculatedto be from 100% to 25%, “+” if efficacy was calculated to be less than25%, and “−” if no data was available. It should be noted that 100%efficacy is the maximum response obtained with4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol.

TABLE 3 Compound Activity % No. EC ₅₀ (μm) Efficacy 1 +++ ++ 2 +++ +++ 3+++ ++ 4 +++ ++ 5 +++ ++ 6 +++ ++ 7 +++ ++ 8 ++ ++ 9 +++ ++ 10 + ++ 11+++ ++ 12 ++ ++ 13 ++ ++ 14 +++ ++ 15 +++ ++ 16 +++ ++ 17 +++ ++ 18 +++++ 19 +++ ++

What is claimed is:
 1. A compound of formula IVA:

or a pharmaceutically acceptable salt thereof, wherein: W is a bond oris an optionally substituted C₁-C₆ alkylidene chain wherein up to twomethylene units of W are optionally and independently replaced by 0,—CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—,—NR′CONR′—, —C(O)NR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—, —SO,—SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—; R^(W) is independentlyR′, halo, cyano, CF₃, CHF₂, OCHF₂, Me, Et, CH(Me)₂, CHMeEt, n-propyl,t-butyl, OH, OMe, OEt, OPh, O-fluorophenyl, O-difluorophenyl,O-methoxyphenyl, O-tolyl, O-benzyl, SMe, SCF₃, SCHF₂, SEt, CH₂CN, NH₂,NHMe, N(Me)₂, NHEt, N(Et)₂, C(O)CH₃, C(O)Ph, C(O)NH₂, SPh,SO₂-(amino-pyridyl), SO₂NH₂, SO₂Ph, SO₂NHPh, SO₂—N-morpholino,SO₂—N-pyrrolidyl, N-pyrrolyl, 2-methylpyrrolyl, 3-fluoropyrrolyl,3,3-difluoropyrrolyl, 3,3-dimethylpyrrolyl, 2,5-dimethylpyrrolyl,N-morpholino, 1-piperidyl, phenyl, benzyl,(cyclohexyl-methylamino)methyl, 4-Methyl-2,4-dihydro-pyrazol-3-one-2-yl,benzimidazol-2yl, furan-2-yl, 4-methyl-4H-[1,2,4]triazol-3-yl,3-(4′-chlorophenyl)-[1,2,4]oxadiazol-5-yl, NHC(O)Me, NHC(O)Et, NHC(O)Ph,or NHSO₂Me; Z is —CH—, —CR¹—, or N, m is 0-5; k is 0-1; each of R¹ isindependently H, C1-C4 aliphatic, CF₃, halo, or C3-C6 cycloaliphatic; R²is hydrogen; R³ is hydrogen; R⁴ is hydrogen or a C₁₋₆ aliphatic; R′ isindependently selected from hydrogen or an optionally substituted groupselected from a C₁-C₈ aliphatic group, a 3-8-membered saturated,partially unsaturated, or fully unsaturated monocyclic ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-12 membered saturated, partially unsaturated, or fully unsaturatedbicyclic ring system having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; or two occurrences of R′ are taken togetherwith the atom(s) to which they are bound to form an optionallysubstituted 3-12 membered saturated, partially unsaturated, or fullyunsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 2. The compoundaccording to claim 1, wherein R² and R⁴ is hydrogen.
 3. The compoundaccording to claim 1, wherein R¹ is hydrogen.
 4. The compound accordingto claim 1, wherein R¹ is C1-C3 alkyl.
 5. The compound according toclaim 4, wherein R¹ is methyl.
 6. The compound according to claim 4,wherein R¹ is ethyl.
 7. The compound according to claim 1, wherein eachoccurrence of WR^(W) is independently —C1-C3 alkyl, t-butyl, C1-C3perhaloalkyl, —OH, —O(C1-C3alkyl), —CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or—COOR′, —COR′, —O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′),—(CH₂)₂OR′, —(CH₂)OR′, optionally substituted 5-7 membered heterocylicring, optionally substituted 5-7 membered cycloaliphatic group,optionally substituted monocyclic or bicyclic aromatic ring, optionallysubstituted arylsulfone, optionally substituted 5-membered heteroarylring, —N(R′)(R′), —(CH₂)₂N(R′)(R′), —C≡—CCH₂N(R′)(R′) or—(CH₂)N(R′)(R′).
 8. The compound according to claim 1, wherein saidcompound has formula VA-1:

wherein each of WR^(W2) and WR^(W4) is independently selected fromhydrogen, CN, CF₃, OCF₃, halo, C1-C6 straight or branched alkyl, 3-12membered cycloaliphatic, phenyl, C5-C10 heteroaryl or C3-C7heterocyclic, wherein said heteroaryl or heterocyclic has up to 3heteroatoms selected from O, S, or N, wherein said WR^(W2) and WR^(W4)is independently and optionally substituted with up to threesubstituents selected from —OR′, —CF₃, —OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃,halo, CN, —COOR′, —COR′, —O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′),—CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, CH₂CN, optionally substitutedphenyl or phenoxy, —N(R′)(R′), —NR′C(O)OR′, —NR′C(O)R′,—(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′); and WR^(W5) is selected fromhydrogen, halo, —OH, NH₂, CN, CHF₂, NHR′, N(R′)₂, —NHC(O)R′, —NHC(O)OR′,NHSO₂R′, —OR′, CH₂OH, CH₂N(R′)₂, C(O)OR′, C(O)N(R′)₂, SO₂NHR′,SO₂N(R′)₂, OSO₂N(R′)₂, OSO₂CF₃, or CH₂NHC(O)OR′.
 9. The compoundaccording to claim 1, wherein said compound has the formula VA-2:

wherein: ring B is a 5-7 membered monocyclic or bicyclic, heterocyclicor heteroaryl ring optionally substituted with up to n occurrences of-Q-R^(Q), Q is W; R^(Q) is R^(W); m is 0-4; and n is 0-4.
 10. Thecompound according to claim 1, wherein said compound has the formulaVA-3:

wherein: Q is W; R^(Q) is R^(W); m is 0-4; and n is 0-4.
 11. Thecompound according to claim 1, wherein said compound is selected fromN-(2,4-di-tert-butyl-5-hydroxyphenyl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(4-(3,3-dimethylpyrrolidin-1-yl)-2-(trifluoromethyl)phenyl)-7-methyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,N-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,7-ethyl-N-(2-fluoro-5-hydroxy-4-(1-methylcyclohexyl)phenyl)-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,7-oxo-N-(4-(pyrrolidin-1-yl)-2-(trifluoromethyl)phenyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(2-fluoro-5-hydroxy-4-(1-methylcyclohexyl)phenyl)-7-methyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,N-(4-cyclopentyl-5-hydroxy-2-(trifluoromethyl)phenyl)-7-methyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,N-(2,4-di-tert-butyl-5-hydroxyphenyl)-7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,N-(4-cyclopentyl-5-hydroxy-2-methylphenyl)-7-methyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamideorN-(2-cyano-4-cyclopentyl-5-hydroxyphenyl)-7-methyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide.12. A pharmaceutical composition comprising a compound of formula IVAaccording to claim 1 and a pharmaceutically acceptable carrier oradjuvant.
 13. The composition according to claim 12, wherein saidcomposition comprises an additional agent selected from a CFTRmodulator.
 14. A method of modulating CFTR activity comprising the stepof contacting said CFTR with a compound of formula IVA according toclaim
 1. 15. A method of treating or lessening the severity of a diseasein a patient, wherein said disease is selected from cystic fibrosis,asthma, COPD, or dry-eye disease, said method comprising the step ofadministering to said patient an effective amount of a compound offormula IVA according to claim
 1. 16. A compound of formula IVB, orformula IVC:

or a pharmaceutically acceptable salt thereof, wherein ring A₂ isselected from:

wherein ring A₂ is fused to ring A₁ through two adjacent ring atoms; Wis a bond or is an optionally substituted C₁-C₆ alkylidene chain whereinup to two methylene units of W are optionally and independently replacedby O, —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—, —CO₂—, —OCO—, —NR′CO₂—,—O—, —NR′CONR′—, —C(O)NR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—, —NR′CO—, —S—,—SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—; R^(W) isindependently R′, halo, cyano, CF₃, CHF₂, OCHF₂, Me, Et, CH(Me)₂,CHMeEt, n-propyl, t-butyl, OH, OMe, OEt, OPh, O-fluorophenyl,O-difluorophenyl, O-methoxyphenyl, O-tolyl, O-benzyl, SMe, SCF₃, SCHF₂,SEt, CH₂CN, NH₂, NHMe, N(Me)₂, NHEt, N(Et)₂, C(O)CH₃, C(O)Ph, C(O)NH₂,SPh, SO₂-(amino-pyridyl), SO₂NH₂, SO₂Ph, SO₂NHPh, SO₂—N-morpholino,SO₂—N-pyrrolidyl, N-pyrrolyl, 2-methylpyrrolyl, 3-fluoropyrrolyl,3,3-difluoropyrrolyl, 3,3-dimethylpyrrolyl, 2,5-dimethylpyrrolyl,N-morpholino, 1-piperidyl, phenyl, benzyl,(cyclohexyl-methylamino)methyl, 4-Methyl-2,4-dihydro-pyrazol-3-one-2-yl,benzimidazol-2yl, furan-2-yl, 4-methyl-4H-[1,2,4]triazol-3-yl,3-(4′-chlorophenyl)-[1,2,4]oxadiazol-5-yl, NHC(O)Me, NHC(O)Et, NHC(O)Ph,or NHSO₂Me; Z is —CH—, —CR¹—, or N, m is 0-5; k is 0-1; each of R¹ isindependently H, C1-C4 aliphatic, CF₃, halo, or C3-C6 cycloaliphatic; R²is hydrogen; R³ is hydrogen; R⁴ is hydrogen or a C₁₋₆ aliphatic group;R′ is independently selected from hydrogen or an optionally substitutedgroup selected from a C₁-C₈ aliphatic group, a 3-8-membered saturated,partially unsaturated, or fully unsaturated monocyclic ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-12 membered saturated, partially unsaturated, or fully unsaturatedbicyclic ring system having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; or two occurrences of R′ are taken togetherwith the atom(s) to which they are bound to form an optionallysubstituted 3-12 membered saturated, partially unsaturated, or fullyunsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 17. Thecompound according to claim 16, wherein R² and R⁴ is hydrogen.
 18. Thecompound according to claim 16, wherein R¹ is hydrogen.
 19. The compoundaccording to claim 16, wherein R¹ is C1-C3 alkyl.
 20. The compoundaccording to claim 19, wherein R¹ is methyl.
 21. The compound accordingto claim 19, wherein R¹ is ethyl.
 22. The compound according to claim16, wherein each occurrence of WR^(W) is independently —C1-C3 alkyl,t-butyl, C1-C3 perhaloalkyl, —OH, —O(C1-C3alkyl), —CF₃, —OCF₃, —SCF₃,—F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′),—CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionally substituted 5-7 memberedheterocylic ring, optionally substituted 5-7 membered cycloaliphaticgroup, optionally substituted monocyclic or bicyclic aromatic ring,optionally substituted arylsulfone, optionally substituted 5-memberedheteroaryl ring, —N(R′)(R′), —(CH₂)₂N(R′)(R′), —C≡CCH₂N(R′)(R′) or—(CH₂)N(R′)(R′).
 23. The compound according to claim 16, wherein ringA₁, taken together with ring A₂ in compounds of formula IVB, or formulaIVC is an optionally substituted ring selected from:


24. The compound according to claim to claim 16, wherein said compoundhas the formula VB-1:

wherein: R^(W1) is hydrogen or C1-C6 aliphatic; each of R^(W3) ishydrogen or C1-C6 aliphatic; or optionally both R^(W3) taken togetherform a C3-C6 cycloalkyl or heterocyclic ring having up to twoheteroatoms selected from O, S, or NR′, wherein said ring is optionallysubstituted with up to two WR^(W) substituents; and m is 0-4.
 25. Thecompound according to claim 24, wherein WR^(W1) is hydrogen, C1-C6aliphatic, C(O)C1-C6 aliphatic, or C(O)OC1-C6 aliphatic.
 26. Thecompound according to claim 24, wherein each R^(W3) is hydrogen, C1-C4alkyl; or both R^(W3) taken together form a C3-C6 cycloaliphatic ring or5-7 membered heterocyclic ring having up to two heteroatoms selectedfrom O, S, or N, wherein said cycloaliphatic or heterocyclic ring isoptionally substituted with up to three substitutents selected fromWR^(W1).
 27. The compound according to claim 16 wherein ring A₂ isselected from:

wherein said ring is optionally substituted.
 28. The compound accordingto claim 27, wherein said compound has the formula VB-3:

wherein: G₄ is hydrogen, halo, CN, CF₃, CHF₂, CH₂F, optionallysubstituted C1-C6 aliphatic, aryl-C1-C6 alkyl, or a phenyl, wherein G₄is optionally substituted with up to 4 WR^(W) substituents; wherein upto two methylene units of said C1-C6 aliphatic or C1-C6 alkyl isoptionally replaced with —CO—, —CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—,—NR′CONR′—, —OCONR′—, —NR′CO—, —S—, —NR′—, —SO₂NR′—, NR′SO₂—, or—NR′SO₂NR′—; G₅ is hydrogen, CN, or an optionally substituted C1-C6aliphatic; wherein said indole ring system is further optionallysubstituted with up to 3 substituents independently selected fromWR^(W).
 29. The compound according to claim 28, wherein G₄ is hydrogen,and G₅ is C1-C6 aliphatic, wherein said aliphatic is optionallysubstituted with C1-C6 alkyl, halo, cyano, or CF₃, and wherein up to twomethylene units of said C1-C6 aliphatic or C1-C6 alkyl is optionallyreplaced with —CO—, —CONR′—, —CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—,—OCONR′—, —NR′CO—, —S—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—. 30.The compound according to claim 29, wherein G₄ is hydrogen, and G₅ iscyano, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl,cyanomethyl, methoxyethyl, CH₂C(O)OMe, (CH₂)₂—NHC(O)O-tert-But, orcyclopentyl.
 31. The compound according to claim 29, wherein G₅ ishydrogen, CN or CF₃, and G₄ is halo, C1-C6 aliphatic or phenyl, whereinsaid aliphatic or phenyl is optionally substituted with C1-C6 alkyl,halo, cyano, or CF₃, wherein up to two methylene units of said C1-C6aliphatic or C1-C6 alkyl is optionally replaced with —CO—, —CONR′—,—CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′CO—, —S—, —NR′—,—SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—.
 32. The compound according to claim31 wherein G₅ is hydrogen, CN or CF₃, and G₄ is halo, ethoxycarbonyl,t-butyl, 2-methoxyphenyl, 2-ethoxyphenyl, (4-C(O)NH(CH₂)₂—NMe₂)-phenyl,2-methoxy-4-chloro-phenyl, pyridine-3-yl, 4-isopropylphenyl,2,6-dimethoxyphenyl, sec-butylaminocarbonyl, ethyl, t-butyl, orpiperidin-1-ylcarbonyl.
 33. The compound according to claim 16, whereinsaid compound is selected fromN-(3-tert-butyl-1H-indol-6-yl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(5-tert-butyl-1H-indol-6-yl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(5-tert-butyl-1H-indol-6-yl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,7-ethyl-4-oxo-N-(3-(trifluoromethyl)-1H-indol-6-yl)-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,7-oxo-N-(5-(trifluoromethyl)-1H-indol-6-yl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(1H-indol-6-yl)-2-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxamide,N-(3-tert-butyl-1H-indol-6-yl)-7-ethyl-4-oxo-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,7-ethyl-4-oxo-N-(5-(trifluoromethyl)-1H-indol-6-yl)-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide,or7-methyl-4-oxo-N-(3-(trifluoromethyl)-1H-indol-6-yl)-1,4-dihydropyrrolo[1,2-a]pyrimidine-3-carboxamide.34. A pharmaceutical composition comprising a compound of formula IVB,or formula IVC according to claim 16 and a pharmaceutically acceptablecarrier or adjuvant.
 35. The composition according to claim 34, whereinsaid composition comprises an additional agent selected from a CFTRmodulator.
 36. A method of modulating CFTR activity comprising the stepof contacting said CFTR with a compound of formula IVB, or formula IVCaccording to claim
 16. 37. A method of treating or lessening theseverity of a disease in a patient, wherein said disease is selectedfrom cystic fibrosis, asthma, hereditary emphysema, COPD, or dry-eyedisease, said method comprising the step of administering to saidpatient an effective amount of a compound of formula IVB, or formula IVCaccording to claim 16.